Morphological transformation of Syrian hamster embryo cells by pseudorabies virus related growth factor.

Syrian hamster embryo (SHE) clonal morphological transformation assay has been used for the in vitro testing of transforming activity of Pseudorabies virus (PRV) related growth factor (PRGF). It was found that PRGF induces morphologically transformed colonies of SHE cells in the wide titer scale (in the range 1 x 10(7) to 1 x 10(13) U/ml). The concentrations of PRGF which induced the transforming phenotype of SHE cells in the colonies did not cause significant cytotoxic effect.

Application of porous carbon for solid-phase extraction of dicarboxyimide fungicide residues from wines in combination with high-resolution capillary gas chromatography and gas chromatography-mass spectrometry.

Solid-phase extraction with a novel porous carbon sorbent CARB GR was used for the clean-up step of dicarboxyimide fungicides residues from variety of Slovak grape wines with subsequent capillary gas chromatography-flame ionization detection, -electron-capture detection (ECD) and -mass spectrometry-ion-trap detection (MS-ITD) analysis. Recovery was tested at various concentration levels of vinclozolin and iprodione in standard solutions (R = 80-97%, R.S.D. < or = 5). The value of recovery in spiked wines is dependent on concentration level (studied in the range of 5.9 micrograms/1-1.96 mg/l) and on the variety of wine (R = 80-96%; R.S.D. = 3-5%). Limits of quantitation (for sample volume 50 ml) were determined to be with GC-ECD for both fungicides in ppt range and with GC-MS-ITD in the multiple ion detection mode monitoring in ptt range for vinclozolin and ppb range for iprodione. Concentration levels of vinclozolin residues were determined in treated wines (with 0.1% Ronilan 50 WP) as well as iprodione residues (with 0.15% Rovral 50 WP) and a strong dependence on the protective term before the harvest is shown.

Isotachophoretic determination of naproxen in the presence of its metabolite in human serum.

An isotachophoretic method with conductivity detection was developed to determine naproxen in the presence of its metabolite 6-O-desmethylnaproxen in human serum. The leading electrolyte contained 10 mM hydrochloric acid, beta-alanine, pH 4.0 and 0.1% methylhydroxypropylcellulose. The terminating electrolyte was 10 mM 2-(N-morpholino)ethanesulfonic acid-tris(hydroxymethyl)aminomethane, pH 6.9, containing 20% (v/v) of ethanol. Naproxen was determined in serum supernatant after simple deproteination of the sample with ethanol. The isotachophoretic results were compared with those obtained by synchronous fluorescence spectrometry.

Factors affecting system clotting in continuous renal replacement therapy: results of a randomized, controlled trial.

BACKGROUND: System clotting and the anticoagulation techniques employed to prevent it are important causes of morbidity in continuous renal replacement therapy (CRRT). Different means have been employed in attempts to prolong system lifespan while minimizing complications. SUBJECTS, MATERIALS AND METHODS: To determine whether augmenting blood flow and flush frequency could reduce clotting frequency, we compared system lifespan in a standard blood flow and saline flush group (125 ml/min and 100 ml once hourly, respectively) to an augmented blood flow and saline flush group (200-250 ml/min and 100 ml twice hourly). A total of 34 patients treated with continuous venovenous hemodialysis were randomized to receive either the standard or augmented regimens in a prospective trial conducted between August 1995 and March 1997. A total of 130 systems were studied. RESULTS: Based on intention-to-treat analysis, there was no difference in time to clot between the two groups. In a multivariate analysis of the outcome, red blood cell and platelet transfusion during CRRT were significantly associated with decreased clotting, and systemic heparin infusion significantly prolonged lifespan of CRRT systems. CONCLUSION: Increasing blood flow and flush frequency does not prevent clotting in CRRT. Since this intervention is more costly than standard treatment, its use cannot be justified.

Enhanced proliferation and progesterone production by porcine granulosa cells cultured with pseudorabies virus growth factor (PRGF).

The objective of this research was to study possible interactions of pseudorabies virus growth factor (PRGF) with ovarian tissue. Granulosa cells isolated from porcine ovaries were cultured as monolayers for 6 days in a control medium without PRGF and in medium supplemented with different doses of this agent. Increased population density and change towards more fibroblastic-like shape of cells cultured with 10(9) I.U PRGF was observed when compared with control culture. The cells divided significantly faster during 6 days of culture under the influence of 10(3), 10(4), 10(5), 10(6), 10(7), 10(8) and 10(9) I.U./ml of PRGF at a dose dependent manner. PRGF in a dose 10(9) I.U. added to cultured cells isolated from small and medium follicles did not influence progesterone secretion . An increase of progesterone secretion under the influence of PRGF in all investigated days of cultures was observed in cells isolated from large preovulatory follicles. The marked increase in progesterone content in PRGF treated culture in doses of 0.5x10(7), 0.5x10(8), 0.5x10(9) I.U. was observed during 4 and 6 days of culture. The rise of progesterone content was not connected with increased number of secretory cells, but with a stimulation of production per cell. PRGF exerted no visible effect on progesterone secretion by granulosa cells from small and medium follicles cultured for 6 days. The presented in vitro data provide evidence for a local action of PRGF in the follicle depending on the stage of follicular development and duration of exposure. Precise relevance of the interaction of PRGF with follicular development requires further study.

Dual effect of pseudorabies virus growth factor (PRGF) displayed on actin cytoskeleton.

Pseudorabies virus growth factor (PRGF) was shown to possess transforming activity as well as transformation repressing activity in in vitro systems. In order to better understand these phenomena we studied actin cytoskeleton and its alterations induced by PRGF using normal human fibroblasts VH-10 and transformed cell line HeLa. For specific detection of filamentous actin cells were stained with phalloidin conjugated with fluorescein isothiocyanate (FITC)-phalloidin. PRGF was applied to VH-10 cells for various length of time from 10 min up to 48 h. The effect was very fast and changes in actin filament composition could be detected already after 10 min. In comparison to untreated cells the staining of treated cells was more diffuse and a number of actin microfilaments in individual stress fibers became reduced. After 30 min thick short actin bundles appeared in the perinuclear region. A 24-h exposure resulted in a large reduction of actin bundles. After additional 24 h a partial restoration of actin cytoskeleton in cells was observed. In transformed HeLa cells PRGF induced opposite process than in normal cells: the number of filamentous actin structures increased. We hypothesise that PRGF may act as a transcription-like factor and may initiate changes in gene expression which consequently result in actin cytoskeleton alterations.

Canine distemper virus replication in cells on microcarriers.

Chick embryo and Vero cells were grown on Gelaspher M gelatin microcarriers in suspension culture. The microcarriers had no adverse effects on cell morphology and growth. Microcarrier cell cultures were used for large-scale production of canine distemper virus. Virus yields (TCID50 per ml) were more than 10-times higher as compared to stationary cell culture.

Pseudorabies virus growth factor can be resolved into two active components.

Pseudorabies virus (PRV) growth factor (PRGF) which induces a transformed phenotype in normal MK-2 cells and represses the transformed phenotype of Hela cells was partially purified and resolved into two components (M(r) < 300 and < 180). Each of the PRGF components retained the transforming activity of the original factor in MK-2 cells but lost its transformation-repressing activity in Hela cells. The latter activity of PRGF could be reconstituted by simultaneous application of its two components. Two monoclonal antibodies against gII glycoprotein of PRV were able to neutralize both PRGF activities, thus supporting the previously suggested hypothesis that the PRV gene for glycoprotein gII might be involved in PRGF synthesis.

Large-scale production of infectious bovine rhinotracheitis virus in cell culture on microcarriers.

Large amounts of infectious bovine rhinotracheitis (IBR) virus was produced in Madin-Darby bovine kidney (MDBK) cells in suspension culture on the microcarriers Gelaspher M. Virus yields (TCID50 per ml) were approximately 10-times higher as compared to classical stationary cell culture.

The glycoprotein B gene and its syn3 locus of herpes simplex virus type 1 are involved in the synthesis of virus-associated growth factor (HSGF-1).

A putative growth factor (HSGF-1) associated with herpes simplex virus type 1 (HSV-1), which is similar to PRGF associated with pseudorabies virus, and/or HSGF-2 associated with HSV-2, was described. Experiments with four syncytial (syn) and four nonsyncytial (syn+) HSV-1 strains showed that the ability of this virus to produce HSGF-1 in infected cells is associated with the syn+ phenotype. Double infection of cells with syn+ and syn strain resulted either in enhancement or complete inhibition of HSGF-1 production, depending on the chosen pair of syn+ and syn strains. The studies with the recombinants between the syn+ strain KOS and syn strain ANGpath in the gene for glycoprotein B (gB) and syn3 locus revealed that the gB gene and its syn3 locus play a role in the HSGF-1 synthesis.

Herpes simplex virus type 2 and pseudorabies virus associated growth factors and their role in the latency in vitro.

A putative herpes simplex virus type 2 (HSV-2) growth factor (HSGF-2) was detected in a crude extract from virus infected mouse embryo cells. This factor, similar to previously described pseudorabies virus (PRV) associated growth factor (PRGF) was shown to have ability to morphologically transform non-transformed cells and to repress the transformed phenotype of transformed cells. Both activities could be neutralized with two, out of seven monoclonal antibodies directed against glycoprotein B of HSV-2. Both PRGF and HSGF-2 were detected in human embryo lung cells latently infected with PRV or HSV-2 either at 41 degrees C, or in the presence of phosphonoacetic acid. Human alpha-2 interferon, when present in medium of latently infected cells enhanced the production of both HSGF and PRGF. On the contrary, when latently infected cells were treated with 5-azacytidine the synthesis of both PRGF and HSGF-2 was completely blocked and the virus reactivated from latency replicated to higher titers than in non-treated cells. The role of PRGF and HSGF-2 in the establishment, maintenance and reactivation of latency, as well as in cellular transformation is discussed.

A simple novel procedure for preparation of herpes simplex virus subunit vaccine.

A simple procedure for preparation of herpes simplex virus type 1 (HSV-1) subunit vaccine is described. The method is based on treatment of virus-infected cells with a nonionic detergent, removal of cell nuclei by low speed centrifugation, separation of viral nucleocapsids by high speed centrifugation through a sucrose cushion and, finally, precipitation of viral and cellular glycoproteins and proteins with ammonium sulphate (AS). Unlike to acetone precipitation, affinity chromatography on lectins or hydroxylapatite chromatography, AS precipitation repeatedly yielded the best and most reliable results as judged by relative protein content, antigenicity and immunogenicity of the final vaccine product.

Determination of the antigenic variability of pseudorabies virus field isolates with monoclonal antibodies.

Five stable hybridoma cell lines secreting monoclonal antibodies (MAbs) were obtained by fusing spleen cells of pseudorabies virus (strain BUK) immunized BALB/c mice with mouse myeloma cell line SP2/0. Plate-trapped and antibody-trapped antigen ELISAs were done to compare the interaction of MAbs with TOP and "K" strains and 7 Slovak field isolates. Only 2 Slovak isolates reacted with all the MAbs, 3 MAbs reacted with TOP and "K" strains. One Slovak isolate gave no reaction with any MAbs.

[Problems of hemorrhage in the cerebellum]. / Niekol'ko poznámok k problematike krvácaní do mozocka.

The authors give an account on their experience with the treatment of five patients (three women and two men) with non-traumatic haemorrhage into the cerebellum. The oldest patient was 67 years, the youngest 14 years, the mean age was 51.2 years. In four the cause of haemorrhage was hypertension, in one patient an A-V malformation. The correct diagnosis was established by CT examination, while originally it was assumed that a supratentorial cerebrovascular attack was involved. One female patient recovered after conservative treatment, in the remainder the haematoma was removed by suboccipital craniectomy. One patient died, the fourteen-year-old female patient which was admitted with symptoms of decerebration, survives with a marked neurological deficit, three patients feel well. In one instance hydrocephalus developed which was treated by ventriculo-peritoneal drainage with a valve.